The present invention is generally in the field of modulators of apoptopic cell death and uses thereof in therapeutic applications to inhibit or to enhance apoptosis, as desired depending on the disease and whether or not it is desired to kill the diseased cells or to rescue the diseased cells from apoptopic cell death. Specifically, the present invention concerns novel genes encoding novel proteins belonging to the leucine zipper family, which are capable of inhibiting apoptosis mediated by the CD3/TCR system or by the Fas/Fas-L system, and which are also capable of stimulating lymphocyte activation.
In particular, the present invention concerns a new protein and the gene encoding therefor called GILR, preparation and uses thereof, as well as any isoforms, analogs, fragments and derivatives of GILR, their preparation and uses.
Apoptosis (programmed cell death) is an important intracellular process having an important role in normal cell and tissue development as well as in the control of neoplastic growth (Cohen, 1993; Osborne and Schwartz, 1994; Wyllie et al., 1980; Kerr et al., 1972; Bursch et al., 1992).
A number of stimuli can either induce or inhibit programmed cell death through activation of molecules, involved in the signaling and execution of apoptosis, acting at different levels including the cell membrane, cytoplasm and nucleus. Among these, of note are those intracellular molecules, including some transcription factors, that have been shown to regulate cell growth. In particular, leucine zipper family proteins, such as for instance MYC, FOS and JUN, can modulate cell death (Shi et al., 1992; Smeyne et al., 1993; Goldstone and Lavin, 1994).
Apoptosis is also important in T-cell development (Dent et al., 1990; Ju et al., 1995; MacDonald and Lees, 1990). In particular, negative selection is due to apoptosis activated through the antigen (Ag) interaction with the T-cell-receptor(TCR)/CD3 complex (Smith et al., 1989). Engagement of the TCR/CD3 complex, either by APCs presenting antigenic peptide or by anti-CD3 antibody, triggers a series of activation events, such as for example, the expression of the Fas/Fas-Ligand (Fas/Fas-L) system, that can induce apoptosis in thymocytes, mature T cells and T cell hybridoma (Alderson et al., 1995; Dhein et al., 1995; Ju et al., 1995; Jenkinson et al., 1989; Webb et al., 1990; Yang et al., 1995). For example, triggering of such activation events in T cell hybridomas leads to cell cycle arrest, followed by apoptosis. This activation-induced cell death (AICD, Kabelitz et al., 1993) requires the interaction of Fas with Fas-L (Alderson et al., 1995; Itoh et al., 1991; Yang et al., 1995).
It has been shown that other stimuli, such as cytokines and glucocorticoid hormones (GCH), are also critical regulators of T-cell development (Migliorati et al., 1993; Nieto et al., 1990; Nieto and Lopez-Rivas, 1989; Cohen and Duke, 1984; Wyllie, 1980). For example, dexamethasone (DEX), a synthetic GCH which by itself induces apoptosis in T cell hybridomas and in normal T lymphocytes, can inhibit AICD induced by triggering of the TCR/CD3 complex (Zacharchuk et al., 1990). This inhibition may be due to prevention of activation induced expression of Fas and Fas-L (Yang et al., 1995).
With respect to the above noted Fas/Fas-L system, it should be noted that Fas has also been called the FAS receptor or FAS-R as well as CD95. For simplicity, this receptor will be called xe2x80x98Fasxe2x80x99 herein throughout and its ligand, as noted above, will be called xe2x80x98Fas-Lxe2x80x99 herein throughout.
Fas is a member of the TNF/NGF superfamily of receptors and it shares homology with a number of cell-surface receptors including the p55-TNF receptor and the NGF receptor (see for example Boldin et al., 1995a and 1995b). Fas mediates cell death by apoptosis (Itoh et al. 1991) and appears to act as a negative selector of autoreactive T cells, i.e. during maturation of T-cells, Fas mediates the apoptopic death of T cells recognizing self-antigens. Mutations in the Fas gene, such as the lpr mutations in mice, have been shown to be responsible for a lymphoproliferation disorder in mice resembling the human autoimmune disease, systemic lupus erythomatosus (SLE; Watanabe-Fukunaga et al., 1992). The Fas-L molecule is apparently a cell surface-associated molecule carried by, amongst others, killer T cells (or cytotoxic T lymphocytesxe2x80x94CTLs), and hence, when such CTLs contact cells carrying Fas, they are capable of inducing apoptopic cell death of the Fas-carrying cells. Further, a monoclonal antibody specific to Fas has been prepared which is capable of inducing apoptopic cell death in cells carrying Fas, including mouse cells transformed by cDNA encoding human Fas (see, for example, Itoh et al, 1991).
While some of the cytotoxic effects of lymphocytes are mediated by interaction of lymphocyte-produced Fas-L with the widely-occurring Fas, it has also been found that various other normal cells besides T lymphocytes, express Fas on their surface and can be killed by the triggering of this receptor. Uncontrolled induction of such a killing process is suspected to contribute to tissue damage in certain diseases, for example, the destruction of liver cells in acute hepatitis. Accordingly, finding ways to restrain the cytotoxic activity of Fas may have therapeutic potential.
Conversely, since it has also been found that certain malignant cells and HIV-infected cells carry Fas on their surface, antibodies against Fas, or Fas-L itself, may be used to trigger Fas-mediated cytotoxic effects in these cells and thereby provide a means for combating such malignant cells or HIV-infected cells (see, for example, Itoh et al. 1991). Finding yet other ways for enhancing the cytotoxic activity of Fas may therefore also have therapeutic potential.
As noted above, Fas is related to one of the TNF receptors, namely, the p55-TNF receptor. TNF (both TNF-xcex1 and TNF-xcex2, and as used throughout, xe2x80x98TNFxe2x80x99 will refer to both) has many effects on cells (see, for example, Wallach, D. (1986) In: Interferon 7 (Ion Gresser ed.), p. 83-122, Academic Press, London; and Beutler and Cerami (1987)).
TNF exerts its effects by binding to its receptors, the p55-TNF receptor and the p75-TNF receptor. Some of the TNF-induced effects are beneficial to the organism, such as, for example, destruction of tumor cells and virus-infected cells, and augmentation of antibacterial activities of granulocytes. In this way TNF contributes to the defense of the organism against tumors and infectious agents and contributes to recovery from injury.
Thus, TNF can be used as an anti-tumor and anti-infectious agent.
However, TNF can also have deleterious effects. For example, overproduction of TNF can have a pathogenic role in several diseases, including amongst others, septic shock (Tracey et al., 1986); excessive weight loss (cachexia); tissue damage in rheumatic diseases (Beutler and Cerami, 1987); tissue damage in graft-versus-host reactions (Piquet et al., 1987); and tissue damage in inflammation, to name but a few of the pathogenic effects of TNF.
The above cytocidal effects of TNF is mediated mainly by the p55-TNF receptor in most cells studied so far, which activity is dependent on the integrity of the intracellular domain of this receptor (see, for example, Brakebusch et al., 1992; Tartaglia et al., 1993). In addition, mutational studies indicate that the related Fas and p55 TNF-receptor mediate intracellular signaling processes, ultimately resulting in cell death, via distinct regions within their intracellular domains (see also, for example, Itoh and Nagata, 1993). These regions also designated xe2x80x98death domainsxe2x80x99 present in both these receptors, have sequence similarity. The xe2x80x9cdeath domainsxe2x80x9d of Fas and p55-TNF receptor are capable of self-association, which is apparently important for promoting the receptor aggregation necessary for initiating intracellular signaling (see, for example, Song et al. 1994; Wallach et al., 1994; Boldin et al., 1995a, b), and which, at high levels of receptor expression, can result in the triggering of ligand-independent signaling (Boldin et al., 1995a, b).
Recent studies have indicated that the cytotoxic effects mediated by Fas and p55-TNF receptor involves an intracellular signaling pathway which includes a number of protein-protein interactions, leading from the initial ligand-receptor binding to the eventual activation of enzymatic effector functions, and which include non-enzymatic protein-protein interactions which are involved in the initiation of the signaling for cell death (see also, for example, Nagata and Golstein, 1995; Vandenabeele et al., 1995; and Boldin et al., 1995a, b). Apparently the binding of the trimeric Fas-L and TNF to their receptors results in the interaction of the intracellular domains of these receptors which is augmented by a propensity of the death-domain regions or motifs to self-associate (Boldin et al., 1995a, b), and induced binding of at least two other cytoplasmatic proteins (which can also bind to each other) to the intracellular domains of these receptors, namely, the protein MORT-1 (also called FADD) which binds to Fas (see Boldin et al., 1995b; Chinnaiyan et al., 1995; Kischkel et al., 1995), and the protein TRADD which binds to the p55-TNF receptor (see Hsu et al., 1995; Hsu et al., 1996). A third such intracellular protein has also been identified called RIP (see Stanger et al., 1995) which binds to the intracellular domains of both Fas and the p55-TNF receptor. RIP can also interact with TRADD and MORT-1. Thus, these three intracellular proteins allow for a functional xe2x80x9ccross-talkxe2x80x9d between Fas and the p55-TNF receptor. The interactions between these receptors and their associated proteins (MORT-1, TRADD, RIP) occurs through the xe2x80x98death domainxe2x80x99 motifs present in each of these receptors and proteins.
Thus, the xe2x80x9cdeath domainxe2x80x9d motifs of the p55-TNF receptor and Fas as well as their three associated proteins MORT-1, RIP and TRADD appear to be the sites of protein-protein interactions. The three proteins MORT-1, RIP and TRADD interact with the p55-TNF receptor and Fas intracellular domains by the binding of their death domains to those of the receptors, and for both RIP and TRADD their death domains also self-associate, although MORT-1 differs in this respect in that its death domain does not self-associate. Accordingly, it would seem that the interaction between the three proteins MORT-1, RIP and TRADD is an important part of the overall modulation of the intracellular signaling mediated by these proteins. Interference of the interaction between these three intracellular proteins will result in modulation of the effects caused by this interaction. For example, inhibition of TRADD binding to MORT-1 may modulate the Fas-p55 TNF-receptor interaction. Likewise, inhibition of RIP in addition to the above inhibition of TRADD binding to MORT-1 may further modulate Fas-p55 TNF-receptor interaction.
Recent studies have also implicated a group of cytoplasmatic thiol proteases which are structurally related to the Caenorhabditis elegans protease CED3 and to the mammalian interleukin-1xcex2 converting enzyme (ICE) in the onset of various physiological cell death processes (reviewed in Kumar, 1995 and Henkart, 1996). There have also been some indications that protease(s) of this family may take part in the cell-cytotoxicity induced by Fas and TNF receptors. Specific peptide inhibitors of the proteases and two virus-encoded proteins that block their function, the cowpox protein crmA and the Baculovirus p35 protein, were found to provide protection to cells against this cell-cytotoxicity (Enari et al., 1995; Los et al., 1995; Tewari et al., 1995; Xue et al., 1995; Beidler et al., 1995). Rapid cleavage of certain specific cellular proteins, apparently mediated by protease(s) of the CED3/ICE family, could be demonstrated in cells shortly after stimulation of Fas or TNF receptors (both the p55-TNF receptor and the p75-TNF receptor).
One such protease and various isoforms thereof (including inhibitory ones), designated MACH which is a MORT-1 binding protein and which serves to modulate the activity of MORT-1 and hence of Fas and p55-TNF receptor, and which may also act independently of MORT-1, has been recently isolated, cloned, characterized, and its possible uses also described, as is set forth in detail in the international application No. PCT/US96/10521, and in a recent publication (Boldin et al., 1996). Another such protease and various isoforms thereof (including inhibitory ones), designated Mch4 has also recently been isolated and characterized (Fernandes-Alnemri et al., 1996; Srinivasula et al., 1996). This Mch4 protein is also a MORT-1 binding protein which serves to modulate the activity of MORT-1 and hence likely also of Fas and p55-TNF receptor and which may also act independently of MORT-1.
Moreover, it has also recently been found that besides the above noted cell cytotoxicity activities and modulation thereof mediated by the various receptors and their binding proteins including Fas, p55-TNF receptor, MORT-1, TRADD, RIP, MACH and Mch4, a number of these receptors and their binding proteins are also involved in the modulation of the activity of the nuclear transcription factor NF-xcexaB, which is a key mediator of cell survival or viability, being responsible for the control of expression of many immune- and inflammatory-response genes. For example, it has been found that TNF-xcex1 can actually stimulate activation of NF-xcexaB and thus TNF-xcex1 is capable of inducing two signals in cells, one eliciting cell death and another that protects cells against death induction by inducing gene expression via NF-xcexaB (see Beg and Baltimore, 1996; Wang et al., 1996; Van Antwerp et al., 1996). A similar dual effect for Fas has also been reported (see reference to this effect as stated in above Van Antwerp et al., 1996). It would therefore appear that there exists a delicate balance between cell death and cell survival upon stimulation of various types of cells with TNF-xcex1 and/or the Fas-L, the ultimate outcome of the stimulation depending on which intracellular pathway is stimulated to a greater extent, the one leading to cell death (usually by apoptosis), or the one leading to cell survival via activation of NF-xcexaB.
In addition, recently there has been further elucidated the possible pathway by which members of the TNF/NGF receptor family activate NF-xcexaB (see Malinin et al., 1997 and the various relevant references set forth therein). Briefly, it arises that several members of the TNF/NGF receptor family are capable of activating NF-xcexaB through a common adaptor protein, Traf2. A newly elucidated protein kinase called NIK (see above Malinin et al., 1997) is capable of binding to Traf2 and of stimulating NF-xcexaB activity. In fact, it was shown (see aforesaid Malinin et al.) that expression in cells of kinase-deficient NIK mutants results in the cells being incapable of having stimulation of NF-xcexaB in a normal endogenous manner and also in the cell having a block in induction of NF-xcexaB activity by TNF, via either the p55-TNF receptor or Fas, and a block in NF-xcexaB induction by TRADD, REP and MORT-1 (which are adaptor proteins that bind these p55-TNF and/or Fas receptors). All of the receptors p55-TNF and p75-TNF receptors and Fas, and their adaptor proteins MORT-1, TRADD and RIP bind directly or indirectly to Traf2, which by its binding ability to NIK apparently modulates the induction of NF-xcexaB.
It has been a long felt need to provide a way for modulating the cellular response to Fas-L and to TNF. For example, in the pathological situations mentioned above, where Fas-L or TNF is overexpressed or otherwise present in excess amounts or where the Fas or, at least, p55-TNF receptor is over-activated or overexpressed, it would be desirable to inhibit the Fas-L or TNF-induced cytocidal effects, which in other situations, e.g. in tumor cells or wound healing applications, it would be desirable to enhance the TNF effect, or in the case of Fas, in tumor cells or HIV-infected cells, it would be desirable to enhance the Fas-mediated effect. To this end, a number of approaches have been attempted directed at the receptors themselves (to enhance or to inhibit their activity or amount, as the case may be) or directed at the signaling pathways, as noted above, in which these receptors or their associated proteins play a role (to enhance or inhibit the activities or amounts of the receptors or their associated proteins, as the case may be).
However, heretofore there has not been elucidated the role of glucocorticoid hormones (GCH) in the regulation of lymphocyte apoptosis, in particular, the role that GCH have in inducing gene expression, the product(s) of which may modulate apoptosis in T cells (and possibly other cells as well), which modulation may be by direct or indirect interaction with, or other means of modulation of, the Fas-mediated or the associated/related p55-TNF receptor-mediated intracellular signaling pathways leading to cell death (by apoptosis in which various proteases as noted above are involved) or leading to cell survival (via induction of NF-xcexaB activation as noted above).
It is an object of the present invention to elucidate the role of glucocorticoid hormones (GCH) in the regulation of lymphocyte apoptosis, in particular, to elucidate the gene(s) or gene products(s) induced by GCH which can modulate apoptosis in T cells or in other cells. Hence, it is an object of the present invention to provide novel gene(s) which are induced by GCH, the product(s) of which can modulate apoptosis in T cells or other cells.
It is another object of the present invention to provide novel proteins, including all isoforms, analogs, fragments or derivatives thereof, which are encoded by the novel GCH-induced gene(s), which proteins, isoforms, analogs, fragments or derivatives can modulate apoptosis in T cells or in other cells. These new proteins, isoforms, analogs, fragments or derivatives may modulate apoptosis by modulating the signaling activity of Fas or the p55-TNF receptor intracellularly in a direct or indirect way, or may modulate apoptosis in an entirely Fas-independent and/or p55-TNF receptor-independent manner, by modulating the activity of other intracellular mediators of apoptosis. It should be understood that the modulation of apoptosis can be an enhancement/augmentation of apoptopic cell death or an inhibition of apoptopic cell death, these being the possible ways of direct modulation of apoptosis, be it via Fas- or p55-TNF-receptor-mediated pathways (inclusive of all the associated proteins/enzymes in these pathways as noted above) or via other pathways involving other intracellular mediators of apoptosis.
Indirect modulation of apoptosis is to be understood as, for example, induction by direct or indirect ways, of cell survival pathways (i.e. induction of NF-xcexaB activation or other such cell survival-related pathways), which cell survival pathways essentially counteract apoptopic pathways.
Another object of the present invention is to provide antagonists (e.g. antibodies, peptides, organic compounds, or even some isoforms, analogs, fragments or derivatives) to the above new proteins, isoforms, analogs, fragments or derivatives thereof, which may be used to inhibit their activity in the intracellular signaling process in which they are involved, and hence to inhibit apoptosis, or conversely, to enhance apoptosis (inhibit cell-survival), as desired and depending on the activity of the protein, isoform, analogs, fragment or derivative, the activity of which is to be inhibited by the antagonist. For example, if a novel protein, isoform, analog, fragment or derivative of the invention is augmentory to apoptosis then such an antagonist would serve to block this augmentory role and ultimately block or reduce cell death via apoptosis. Likewise, if a novel protein, isoform, analog, fragment or derivative of the invention is inhibitory to apoptosis then such an antagonist would serve to block this inhibitory activity and ultimately enhance or augment apoptosis, i.e. result in increased cell death.
A further object of the present invention is to use the novel proteins, isoforms, analog, fragments and derivatives thereof, to isolate and characterize additional proteins or factors which may be involved in GCH-induced modulation of apoptosis (i.e. GCH-induced product(s) of gene expression capable of modulating apoptosis in T cells and other cells), which modulation may be as noted above. For example, other proteins may be isolated which may interact with the novel proteins of the invention and influence their activity, or other receptors or intracellular mediators further upstream or downstream in the signaling process(es) may be isolated with which the novel proteins of the present invention interact and hence in whose function the novel proteins of the invention are also involved.
Moreover, it is an object of the present invention to use the above-mentioned novel proteins, isoforms, analogs, fragments and derivatives as antigens for the preparation of polyclonal and/or monoclonal antibodies thereto. The antibodies, in turn, may be used, for example, for the purification of the new proteins from different sources, such as cell extracts or transformed cell lines. These antibodies may also be used for diagnostic purposes, for example, for identifying disorders related to abnormal functioning of cellular effects induced by GCH and/or mediated by the signaling processes in which the novel proteins of the invention play a role, such as, for example, the apoptopic pathways mediated by Fas and/or the p55-TNF receptor or cell survival pathways involving the induction of NF-xcexaB activation, or any other such pathway in which such GCH-induced product(s) play a role.
A further object of the invention is to provide pharmaceutical compositions comprising the above novel proteins, isoforms, analogs, fragments or derivatives, as well as pharmaceutical compositions comprising the above noted antibodies or other antagonists.
In accordance with the present invention, a new gene and a new protein encoded by this gene have been identified and isolated. The new gene has been designated GILR (for: Glucocorticoid Induced Leucine-zipper family Related gene) which encodes a new member of the leucine zipper family. The designation GILR (Glucocorticoid Induced Leucine-Zipper gene) can be used as well as a synonymous. The mouse GILR protein is a protein of 137 amino acid residues characterized by having four leucine residues (at positions 76, 83, 90 and 97xe2x80x94see FIG. 2 and SEQ ID NO: 2) spanned by 7 amino acids and an asparagine residue (at position 87xe2x80x94see FIG. 2 and SEQ ID NO: 2) within the leucine zipper domain (see FIG. 4). The new GILR gene and the product it encodes, being the new GILR protein, were identified and isolated following dexamethasone (DEX) treatment of cells. DEX is a well known glucocorticoid hormone and hence the GILR gene and GILR protein represent a new glucocorticoid-induced gene and protein, respectively. Further, it appears that the GILR gene is induced in thymocytes and peripheral T cells and is also found to be expressed in normal lymphocytes from the thymus, spleen and lymph nodes. Little or no expression of the GILR gene was detected in other non-lymphoid tissues including brain, kidney and liver.
Using the previously cloned murine GILR as a probe, the human homologue of GILR has been cloned and sequenced (see FIG. 13 and SEQ ID NO 5), demonstrating the high level of conservativity of this sequence.
With respect to the biological activity of the new GILR protein, the experimental results indicate that this protein has at least one important activity being its ability to selectively protect T cells from apoptosis. More specifically, GILR expression selectively protects T cells from apoptosis induced by treatment of the T cells with anti-CD3 monoclonal antibody (mAb) but not by treatment with other apoptopic stimuli. This specific anti-apoptopic effect correlates with the inhibition of Fas and Fas-L expression.
Accordingly, GILR expression may also serve to modulate, albeit indirectly, other intracellular pathways, as noted above, in which Fas is involved, for example, the apoptopic processes common to Fas and the p55-TNF receptor in which their associated proteins and enzymes (e.g. MORT-1, TRADD, RIP, MACH, Mch4) are involved, which ultimately cause cell death by apoptosis. GILR, by specifically inhibiting Fas and Fas-L expression may therefore also inhibit pathways in which Fas acts together with the p55-TNF receptor due to the xe2x80x98cross-talkxe2x80x99 between these receptors mediated by the above proteins which bind to both of these receptors. Thus, while GILR expression may serve to inhibit Fas and Fas-L expression in a direct fashion and to a marked extent, GILR expression may also serve to inhibit the p55-TNF receptor""s intracellular signaling activity, albeit in an indirect way and possibly to a lesser extent. In addition, as noted above, Fas is also involved in induction of NF-xcexaB activation and hence GILR expression which inhibits FAS expression can possibly also serve to reduce this activity of Fas, although with apparently less detrimental effects to the cells, as, primarily the block of Fas-mediated apoptosis would serve to save the cells from cell death to a greater extent than would NF-xcexaB activation save the cells.
In view of the above-mentioned it also arises, for example, that when it is desired to kill cells, e.g. tumor or HIV-infected cells, then it would be desirable to inhibit GILR expression, whilst, conversely, when it is desired to protect cells, e.g. liver cells in hepatitis patients, then it would be desired to increase the expression of GILR or augment its activity. Other uses of GILR and the control of its expression will be set forth herein below in greater detail.
As is detailed herein below, by comparing untreated and DEX-treated cells (for example, murine thymocytes, although any mammalian thymocytes and/or peripheral T cells and/or other lymphocytes, such as those obtained from humans, may be used equally) by employing the subtraction probe technique, it was possible to identify, isolate and clone the new GILR gene of the present invention.
The novel GILR protein of the invention is, in view of the above-mentioned and as set forth herein below, a modulator of apoptosis in lymphocytes, in general, and is in particular apparently an inhibitor of Fas-(and Fas-L-)mediated apoptosis, especially in T-lymphocytes.
Sequencing of the new GILR gene and protein has revealed that these are novel as based on comparisons of the GILR nucleotide and amino acid sequences (see FIGS. 2 and 13) with known sequences in various databases.
These comparisons revealed some homology between these GILR sequences and any known sequences.
The proteins that show the higher degree of homology are hDIP (Vogel et al., 1996) and human TSC-22 (Jay et al., 1996) (FIG. 15). All contain a similar leucine zipper domain (FIG. 4). Both these protein have been poorly characterized as potential transcriptional factor with a widespread distribution among different tissues. In this regard, GILR shows clearly distinct expression profile and activity, as it will be demonstrated in the examples.
In summary, based on the above mentioned and also taking into account the biological properties of the leucine zipper family which GILR is a member, it arises that, in general, GILR may be used to stimulate lymphocyte activity and to rescue cells from apoptotic cell death. GILR may of course, also be used as a probe to isolate other molecules which bind to GILR and which may serve to modulate its activity or otherwise be involved in intracellular signaling processes.
Accordingly, the present invention provides a DNA sequence encoding a glucocorticoid-induced leucine-zipper family related protein (GILR), isoforms, fragments or analogs thereof, said GILR, isoforms, fragments or analogs thereof capable of inhibiting apoptosis and stimulating lymphocyte activity.
Embodiments of the DNA sequence of the invention include:
(a) a cDNA sequence derived from the coding region of a native GILR protein;
(b) DNA sequences capable of hybridization to a sequence of (a) under moderately stringent conditions and which encode a biologically active GILR protein; and
(c) DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a) and (b) and which encode a biologically active GILR protein.
Other embodiments of the above DNA sequence are sequences comprising at least part of the DNA sequence depicted in SEQ ID NO:1 and encoding at least one active GILR protein, isoform, analog or fragment; as well as a DNA sequence encoding a GILR protein, isoform, analog or fragment having at least part of the amino acid sequence depicted in SEQ ID NO: 2.
Further embodiments of the above DNA sequence are sequences comprising at least part of the DNA sequence depicted in SEQ ID NO:5 and encoding at least one active human GILR protein, isoform, analog or fragment; as well as a DNA sequence encoding a human GILR protein, isoform, analog or fragment having at least part of the amino acid sequence depicted in SEQ ID NO: 6.
The present invention also provides a vector comprising any one of the above DNA sequences.
The vectors of the present invention are capable of being expressed in a eukaryotic host cell, or of being expressed in a prokaryotic host cell.
Accordingly, the present invention also provides transformed eukaryotic or prokaryotic host cells containing any of the above vectors.
By another aspect of the invention, there is provided a GILR protein, isoform, fragment, functional analogs or derivatives thereof encoded by any of the above DNA sequences, said protein, isoform, fragment, analogs and derivatives thereof being capable of inhibiting apoptosis and stimulating lymphocyte activity.
Embodiments of the above proteins, etc. of the invention include a GILR protein, isoform, fragment, analogs and derivatives thereof, wherein said protein, isoform, analogs, fragments and derivatives have at least part of the amino acid sequence SEQ ID NO: 2. or of the aminoacid sequence SEQ ID NO: 6.
The present invention also provides for a method for producing the GILR protein, isoform, fragment, analogs or derivatives thereof, comprising growing the transformed host cells under conditions suitable for the expression of said protein, analogs or derivatives, effecting post-translational modifications as necessary for obtaining of said protein, fragments, analogs or derivatives and isolating said expressed protein, fragments, analogs or derivatives.
In another aspect, there is provided antibodies or active fragments or derivatives thereof, specific for the GILR protein, isoform, fragment, analogs or derivatives of the invention.
The above DNA sequences and GILR proteins, etc. encoded thereby of the invention have many possible uses, and accordingly the present invention also provides for the following methods. It must be stressed that other therapeutic uses of GILR, its isoforms, analogs, fragments and derivatives, as well as antibodies against it and other antagonists of GILR activity, e.g. peptides, are also envisioned within the scope of the present invention, as are set forth in the detailed description of the invention or as arise from the disclosure herein below. Accordingly the following are but representative of the various methods in accordance with the present invention:
(i) A method for the inhibition of apoptosis in cells, mediated by the Fas/Fas-L system, CD3/TCR system or other intracellular mediators of apoptosis, comprising treating said cells with one or more GILR proteins, isoforms, analogs, fragments or derivatives, wherein said treating of said cells comprises introducing into said cells said one or more proteins, isoforms, analogs, fragments or derivatives in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more proteins, isoforms, analogs, fragments or derivatives in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.
(ii) A method as in (i) above for the inhibition of apoptosis in cells, wherein said treating of cells comprises introducing into said cells a DNA sequence encoding said GILR protein, isoforms, analogs, fragments or derivatives in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.
(iii) A method as in (i) or (ii) above wherein said treating of said cells is by transfection of said cells with a recombinant animal virus vector comprising the steps of:
(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein (ligand) that is capable of binding to a specific cell surface receptor on the surface of said cells to be treated and a second sequence encoding a protein selected from the GILR protein, isoforms, analogs, fragments and derivatives, that when expressed in said cells is capable of inhibiting apoptosis; and
(b) infecting said cells with said vector of (a).
(iv) A method for enhancing apoptosis in cells by inhibiting the activity if GILR proteins in said cells, comprising treating said cells with antibodies or active fragments or derivatives thereof, said treating being by application of a suitable composition containing said antibodies, active fragments or derivatives thereof to said cells.
(v) A method for enhancing apoptosis in cells by inhibiting the activity of GILR proteins in said cells, comprising treating said cells with an oligonucleotide sequence encoding an antisense sequence for at least part of the DNA sequence encoding a GILR protein, said oligonucleotide sequence being capable of blocking the expression of the GILR protein.
(vi) A method as in (v) above wherein said oligonucleotide sequence is introduced to said cells via a virus of (iii) above wherein said second sequence of said virus encodes said oligonucleotide sequence.
(vii) A method for treating tumor cells or HIV-infected cells or other diseased cells, to enhance apoptosis in said cells by inhibiting the activity of GILR proteins in said cells, comprising:
(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein capable of binding to a specific tumor cell surface receptor or HIV-infected cell surface receptor or receptor carried by other diseased cells and a sequence encoding an inactive GILR mutant protein, said mutant protein, when expressed in said tumor, HIV-infected, or other diseased cell is capable of inhibiting the activity of normal endogenous GILR and enhancing apoptosis in said cells; and
(b) infecting said tumor or HIV-infected cells or other diseased cells with said vector of (a).
(viii) A method for enhancing apoptosis in cells by inhibiting the activity of GILR proteins in said cells, comprising applying the ribozyme procedure in which a vector encoding a ribozyme sequence capable of interacting with a cellular mRNA sequence encoding a GILR protein, is introduced into said cells in a form that permits expression of said ribozyme sequence in said cells, and wherein when said ribozyme sequence is expressed in said cells it interacts with said cellular mRNA sequence and cleaves said mRNA sequence resulting in the inhibition of expression of said GILR protein in said cells.
(ix) A method for enhancing apoptosis in cells by inhibiting the activity of GILR proteins in said cells, comprising introducing into said cells a peptide that is capable of binding the normal endogenous GILR in said cells and inhibiting its activity thereby enhancing apoptosis.
(x) A method for isolating and identifying proteins, which are GILR-like proteins belonging to the leucine zipper family or are proteins capable of binding directly to GILR, comprising applying the yeast two-hybrid procedure in which a sequence encoding said GILR is carried by one hybrid vector and sequence from a cDNA or genomic DNA library is carried by the second hybrid vector, the vectors then being used to transform yeast host cells and the positive transformed cells being isolated, followed by extraction of the said second hybrid vector to obtain a sequence encoding a protein which binds to said GILR.
(xi) A method as in any of the above wherein said protein is at least one of the GILR isoforms, analogs, fragments and derivatives thereof.
By yet another aspect of the present invention there are provided various pharmaceutical compositions, which are particularly useful for effecting at least some of the above methods of the invention. The following is therefore but a representative number of possible pharmaceutical compositions in accordance with the present invention, other possible compositions/formulations within the scope of the present invention are as set forth in the following detailed disclosure or as clearly arising therefrom:
a) a pharmaceutical composition for the inhibition of apoptosis in cells or for stimulating lymphocyte activation, comprising, as active ingredient, at least one GILR protein, its biologically active fragments, analogs, derivatives or mixtures thereof
b) a pharmaceutical composition for inhibiting apoptosis in cells or for stimulating lymphocyte activation comprising, as active ingredient, a recombinant animal virus vector encoding a protein capable of binding a cell surface receptor and encoding at least one GILR protein, isoform, active fragments or analogs.
c) a pharmaceutical composition for enhancing apoptosis in cells by inhibiting GILR activity in said cells, comprising as active ingredient, an oligonucleotide sequence encoding an anti-sense sequence of the GILR protein mRNA sequence.
d) a pharmaceutical composition for enhancing apoptosis in cells by inhibiting GILR activity in said cells, comprising, as active ingredient, an inactive mutant GILR protein or DNA sequence encoding said inactive mutant GILR protein, which GILR mutant, when introduced into, or expressed in, said cells inhibits the activity of the normal endogenous GILR protein.
e) a pharmaceutical composition for enhancing apoptosis in cells by inhibiting GILR activity in said cells, comprising, as active ingredient, a peptide capable of binding to the active site or the leucine zipper domain of GILR and thereby inhibiting normal endogenous GILR activity in cells.